A new era in medical sciences has been generated by the remarkable advances made in the field of genetic engineering. The genetic engineering revolution has been hastened by the discovery of naturally occurring enzymes which cleave double helical deoxyribonucleic acid (hereafter DNA) molecules. These enzymes, called restriction endonucleases, cleave DNA molecules at very specific recognition sites within the DNA polymer. The recognition sites are specific sequences of nucleotides for each restriction enzyme. The sequence-specific cleavage of DNA has found many applications such as DNA sequence determinations, chromosome analyses, gene isolation and recombinant DNA mainipulations. Other applications include new and useful diagnostic reagents to detect pathogens and aberrant DNA molecules.
The usefulness of restriction endonucleases has been limited to cleavage of double-stranded DNA molecules containing the nucleic acid sequences recognized by the limited number of these enzymes. In addition, DNA cleavage by restriction endonucleases is limited to the cleavage of DNA at loci where the sequence recognition site occurs. Thus, endonucleases cannot be used to specifically excise a particular piece of DNA unless, by chance, that piece of DNA contains specific nucleic acid sequences recognized by the limited number of known endonucleases.
The development of synthetic reagents for the sequence-specific modification of DNA provides additional tools useful in research, diagnostics and chemotherapeutic strategies. For example, the attachment of a DNA-cleaving moiety such as ethylenediaminetetraacetic acid-iron complex, (hereafter EDTA-Fe(II)), to a DNA binding molecule produces an efficient DNA cleaving molecule as described by Hertzbert & Dervan, J. Am. Chem. Soc. 104, p. 313-315 (1982); Biochemistry 23, p. 3934-3945 (1984). Methidiumpropyl-EDTA (hereafter MPE), which contains the metal chelator EDTA tethered to the DNA intercalator methidium, has been shown to cleave double helical DNA efficiently in a reaction dependent on ferrous ion (FeII) and dioxygen (O.sub.2). Addition of reducing agents such as dithiothreitol (hereafter DTT) increases the efficiency of DNA cleavage, as reported by Hertzberg & Dervan, J. Am. Chem. Soc. 104, p. 313-315 (1982); Biochemistry 23 p. 3934-3945 (1984). MPE-Fe(II) cleaves DNA in a relatively nonsequence specific manner and with significantly lower sequence specificity than the enzyme DNAse I and is thus useful as a research tool in " footprinting" experiments to identify the binding locations of small molecules such as drugs and proteins on native DNA. Van Dyke & Dervan, Cold Spring Harbor Symp. Quant. Biol. 47, p. 347-353 (1982); Biochemistry 22 p. 2373-2377 (1983); Nucleic Acids Res. 11, p. 5555-5567 (1983); and Science 225 p. 1122-1127 (1984).
Many small molecules important in antibiotic, antiviral and antitumor chemotherapy bind to double helical DNA. Until recently knowledge of the DNA base sequence specificities for these small DNA-binding molecules, such as antibiotics, was limited due to the need to rely on the overall binding affinity of such drugs to homopolymer and copolymer DNAs. The attachment of the cleaving complex EDTA-Fe(II) to antibiotics such as distamycin (hereafter DE) followed by DNA cleavage pattern analyses from Maxam-Gilbert sequencing gels has yielded information on the DNA binding sites and orientation of such drugs on DNA. Hertzerg and Dervan, J. Am. Chem. Soc., 104, p. 313-315 (1982); Taylor et al., Tetrahedron 40, p. 457-465 (1984); Science, 225, p. 1122-1127 (1984).
The mechanism of cleavage by EDTA-FeII complexed with synthetic molecules such as methidium or antibiotics such as distamycin is thought to occur by a common mechanism wherein MPE or DE bind in the minor groove of the right-handed DNA helix by hydropobic and hydrogen binding interactions. Cleavage most likely involves diffusible hydroxyl radical. Hertzberg and Dervan, Biochemistry 23, p. 3934-3945 (1984); Tetrahedron, 40, pg. 457-465 (1984).
Nucleic acid hybridization probes consisting of sequences of deoxyribonucleotides (DNA) or ribonucleotides (RNA) are well-known in the art. Typically, to construct a probe, selected target DNA is obtained as a single strand and copies of a portion of the strand are synthesized in the laboratory and labelled using radioactive isotopes, fluorescing molecules or enzymes that react with a substrate to produce a color change. When exposed to complementary strands of target DNA, for example in a sample of tissue fluid taken from a patient, the labelled DNA probe binds to (hybridizes) its complementary DNA sequence. The label on the probe is then detected and the DNA of interest is thus located. The probe may also be used to target RNA sequences. Finally, probes constructed of RNA sequences may be used to hybridize with a single complementary strand of double-helical DNA forming heteroduplexes without necessitating denaturation of the double-helical DNA. Thomas, et al., Proc. Nat. Acad. Sci. 73, p. 2294-2298 (1976); Casey and Davidson, Nucl. Acids Res., 4, p. 1539-1552 (1977). DNA probes are proving useful in locating and identifying selected genes, and in the diagnosis and treatment of infection, genetic disorders and cancer. See, U.S. Pat. No. 4,358,535.
The above described methods for sequence-specific DNA cleavage have been limited to double-stranded DNA and to those sequences of DNA recognized by antibiotics and DNA intercalators such as methidium. It would provide increased specificity and flexibility with regard to the possible target nucleic acid sequences if sequence-specific cleavage of single stranded nucleic acid (DNA and RNA) and a wider variety of nucleic acid sequences could be accomplished.
Accordingly, it is an object of this invention to provide a method for preparing novel polynucleotide-chelator probes for recognizing specific nucleic acid sequences.
It is another object of this invention to provide a method for using polynucleotide-chelator probes to cleave single-stranded nucleic acid at a specific location.
Yet another object of this invention is to provide a method for using polynucleotide-chelator probes for chemotherapeutic and diagnostic purposes.
Still another object of this invention is to provide polynucleotide-chelator probes containing novel nucleosides functionalized with a metal chelator.
These and other objects and advantages of the invention will be apparent from the detailed description which follows.